| Cordyceps militaris has become the substitute for Cordyceps sinensis due to its extensive biological activity and pharmacological effects. However, with the increasing market demand,the natural resources of Cordyceps militaris was digged indiscriminately and seriously. In order to make up the lacking resources of wild natural Cordyceps militaris, mass production of cultivating became the focus of attention. At present, production or some effective ingredients of Cordyceps militaris was mostly considered as single investigation index in artificial cultivation, but there was not yet an effective evaluation index to simultaneously improve the production of fruit body and several kinds of active constituents content of Cordyceps militaris solid cultivation technology. In this study, the comprehensive value D, which was the mixture of growth period, production, polysaccharide, adenosine, protein and mannitol yield of fruit body., was used as an index to screening mutation by NTG, and optimize cultivation condition of solid culture, combined with Plackett-Burman and Box-Behnken experimental design. At last, MTT method was investigated fruit body polysaccharide antitumor activity in vitro.Cordyceps militaris CGMCC 5.699 was mutanted by NTG. Then the mutants were screened and tested of genetic stability by value D after screening. Cordyceps militaris N165 was obtained, whose growth period reached 39 days, which decreased 13.33%, production of fruit body, polysaccharide, adenosine, protein and mannitol reached 4.18g, 71.21mg/g,559.58 mg/g, 3.94 mg/g,107.83 mg/g, increased by 43.64%, 30.92%, 16.02%, 91.26%,9.15%.In order to optimize the best nutrient solution components in the solid culture medium of N165, the concentration of (NH4)2SO4, sucrose, silkworm chrysalis powder, KH2PO4, MgSO4·7H2O, CaCl2, ZnCl2 and vitamin B1 were investigated by means of Plackett-Burman to determine the major variable affecting the value of D, which were sucrose, silkworm chrysalis powder, MgSO4·7H2O. Then the Box-Behnken was used to determine the optimal concentration of each factor, and the best composition of solid culture was that 40g of rice adding the best nutrient solution 60mL of sucrose 19.71 g/L, silkworm pupa powder is 16.17 g/L, MgSO4·7H2O 5.00 g/L, (NH4)2SO4 10 g/L, KH2PO4 1g/L, CaCl2 1g/L, ZnCl2 0.01 g/L, vitamin B1 0.05 g/L. Based on the best solid medium, the optimal culture conditions was at 15℃, 70% relative humidity in the dark culture lab training, when the mycelium color change was completing, increasing light and temperature difference to stimulate fruiting body primordia formation, with light scattering 600Lux per day, temperature 25℃, relative humidity 90%, ventilation l0 min per day to supplement the fresh air. In addition, the nutrient solution with adding 3mg/L auxin IAA could shorten the growth cycle. Under these conditions, the value of D reached 0.82, growth period decreased to 28 days, the production of fruit body, polysaccharide, protein, adenosine, mannitol reached 6.97g, 92.63g, 672.64g, 8.79g and 178.63g.MTT method was used to detect the inhibition to Hela, HepG-2 and A549 cells in vitro of fruit body polysaccharides in the mutagenic strain and primitive strain. The results showed that the inhibition of two strains'polysaccharides had time-depentent. After 72h, the IC50 of the mutagenic strain and primitive strain fruit body polysacc- harides to Hela were 93.290μg/mL and 105.924μg/mL,to HepG-2 were 98.449μg/mLand 102.150μg/mL, to A549 were 220.125μg/mLand 237.918μg/mL,which showed that the inhibition of mutagenic strain's polysaccharides was same as primitive strain, the inhibition of mutagenic strain's polysaccharides on Hela was stronger than HepG-2 and A549, while the primitive strain on HepG-2 was the strongest, and the inhibition of both two strains on A549 were the smallest. |
